Container for preparing serum and regenerative medical process using the same

ABSTRACT

A blood storage container suitable for quick and efficient production of a large amount of serum while ensuring high safety, and a method of separating blood and a regenerative medical process using the same are provided. A blood component separation storage apparatus is provided for separating a plurality of blood components of blood so as to be stored therein. The blood component separation storage apparatus includes a blood reservoir for holding the blood and a component storage part connected to the blood reservoir aseptically and in an air-tight manner. The blood reservoir contains an anticoagulant which suppresses coagulation of the blood. The blood reservoir has a serum producing function to remove coagulation factors from the blood to an extent enabling use in practical applications as a serum, and the component storage part stores each blood component generated by separation of the blood in the blood reservoir.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of co-pending application Ser. No.10/557,561, filed on Nov. 21, 2005, which is a 371 of PCT/JP04/07310,filed May 21, 2004, for which priority is claimed under 35 U.S.C. §120;and this application claims priority of Application No. 2003-144036filed in Japan on May 21, 2003 under 35 U.S.C. §119, the entire contentsof all of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to blood storage containers, and relatesto methods of separating blood and regenerative medical process usingthe same.

BACKGROUND ART

Currently, in the field of regenerative medicine, studies in which stemcells collected from a subject are caused to proliferate ordifferentiate ex vivo, and are thereafter transplanted into a subject,thereby promoting regeneration of tissue of the subject have beencarried out. Stem cells are multipotent and can differentiate into avariety of tissues and organs, and they have been attracting attentionas cells which are key to regenerative medicine.

It has been known that in ex vivo cultural proliferation of stem cells,addition of a serum to the medium is effective. However, when humantherapies are targeted, use of a serum derived from an animal other thanfrom the human body should be avoided in light of possible problems ofsafety. Therefore, use of a serum prepared from blood which is derivedfrom a human, in particular, which was collected from the same subjecthas been desired. In addition, culture of stem cells in the field ofregenerative medicine requires relatively larger amounts of serum incomparison with blood tests.

As a method of preparing such a serum, a method in which a bloodcollection tube is used that contains a blood coagulation acceleratingsubstance, such as glass powder is disclosed in JP-A No. 2000-000228.

DISCLOSURE OF THE INVENTION

However, the method described in JP-A No. 2000-000228 uses a bloodcollection tube with a low capacity aimed at blood tests, and therefore,preparatory procedures must be repeated many times to prepare a serum inamounts required for culture of stem cells. Hence, this method is notsuited for practical applications.

Additionally, prepared serums are often temporarily preserved bychilling or freezing until use. Hence, a procedure for transferring fromthe blood collection tube to a container for preservation is required.Repetition of such a procedure may increase the probability ofcontamination of the serum by microorganisms. Furthermore, a serumseparating agent is often added to such blood collection tubes,therefore, possible contamination of the serum with an impurity derivedfrom this serum separating agent cannot be reliably avoided.Accordingly, also in terms of safety and hygiene, use of the methoddisclosed in JP-A No. 2000-000228 is not suitable for the field ofregenerative medical for humans. Moreover, according to existing bloodcollection tubes for preparing a serum, recovery of components otherthan the serum is difficult due to the structure of the blood collectiontube, and in addition, recycling thereof is impossible because suchcomponents form clots.

As described above, development of a container for preparing a serum hasbeen desired which is suitable for preparing large amounts of serum forculturing stem cells.

The present invention was achieved in view of the foregoing problems.Thus, an object of the present invention is to provide a blood storagecontainer suitable for quick and efficient production of a large amountof serum while securing high safety, and a method of separating bloodand a regenerative medical process using the same.

In order to accomplish the aforementioned object, the present inventionprovides a blood component separator having a serum producing functionwhich produces serum in practical usable amounts in the presence ofhumoral components and platelets derived from the blood, by acoagulation activating action.

The term “blood” used herein means whole blood including hemocytes(erythrocytes, leucocytes, platelets) and plasma (serum) that is aliquid component, and liquid containing at least one of these (forexample, blood collected by apheresis). Furthermore, the term “serum”means a pale yellow liquid obtained by allowing collected blood tostand, resulting in reduction of the fluidity, followed by separationfrom the red coagulated block (clot). The “serum” according to thepresent invention is different from common serums in terms of theproduction process without including separation from the clot, but itmeans a humoral component in the blood that is useful in cell cultureand that includes coagulation factors and growth factors substantiallyequivalent to those in common serums.

The term “humoral component derived from blood” means “blood componentsother than hemocytes” or “mixture of blood components other thanhemocytes and an agent such as an anticoagulant added thereto”.

The term “coagulation activating action” means activation of coagulationfactors in the blood to eliminate the coagulation factors from theblood.

The term “serum producing function which produces a serum in apractically usable amount” means, for example, an amount which can beused in culturing stem cells. In culturing stem cells, a serum isrequired in an amount of approximately 10% of the medium. Therefore, theamount is preferably from 5 ml and to 1000 ml, and more preferably from10 ml to 600 ml.

Specifically, the following is provided.

The present invention provides a blood component separator, an apparatusfor separating collected blood into a plurality of blood components andstoring them, the apparatus comprising a blood reservoir for holding theblood, having serum producing function to eliminate coagulation factorsfrom the blood in order to prepare a serum in a practical usable amount.

According to the blood component separator described above, collectedblood can be held in large amounts in the blood reservoir by having theblood reservoir. Additionally, because a serum producing function isprovided to this blood reservoir, rapid activation of platelets andcoagulation factors in the blood is made possible. Furthermore, becausethese factors to be activated can be rapidly eliminated, large amountsof serum can be prepared. This blood component separator preferablycomprises a component storage part for storing a plurality of bloodcomponents separated from the blood. It is more preferred that thiscomponent storage part and the blood reservoir be aseptically connectedin an air-tight manner. Accordingly, a series of steps, from collectionof the blood to preparation of the serum, can be carried out withoutexposure to the air outside. Therefore, the risk of contamination bymicroorganisms can be reduced.

The blood component separator according to the present invention canalso be used as a blood component separator for use in nonhuman, becauseit can be used not only for human blood but also for the blood ofmammals such as rodents, livestock, and primates.

In the aforementioned blood component separator, the plurality of bloodcomponents may include a serum, and may include leukocytes anderythrocytes.

According to the present invention, the serum separated by the bloodcomponent separator contains many cell growth factors, and therefore, itis suited for use in the field of regenerative medicine. In addition,because factors to be activated can be separately recovered, effectiveutilization of the collected blood is permitted without discarding thesame.

In the aforementioned blood component separator, the blood reservoir andthe component storage part are flexible bags, and the aforementionedserum producing function may be provided by a blood coagulationaccelerating substance disposed inside the blood reservoir.

The blood reservoir and the component storage part are flexible bagswhich are lightweight and portable. At least one of each is provided,but pluralities thereof also may be provided. It is preferred that oneblood reservoir and two or more component storage parts be provided.Furthermore, each volume is not particularly limited as long as they cansubstantially separate each blood component from the blood, but ispreferably from 5 (ml) to 1000 (ml), and particularly preferably from 10(ml) to 600 (ml). In addition, by providing a blood coagulationaccelerating substance inside this blood reservoir, the serum producingfunction can be imparted. The blood coagulation accelerating substanceis included to an extent to enable removal of the blood coagulationfactors such as fibrin and platelets from the blood. Furthermore,because the blood coagulation accelerating substance is insoluble inblood, the contamination of impurities in the resulting serum can beavoided.

In the blood component separator, it is preferred that the bloodcoagulation accelerating substance be insoluble in the aforementionedblood, and have a block-like shape. Also, it is more preferred that theblood coagulation accelerating substance be provided in a freely movablemanner in the blood reserved in blood reservoir.

According to this invention, handling during manufacture can be improvedby employing the blood coagulation accelerating substance having theshape of particles or granules, or blocks. Additionally, by providingthe blood coagulation accelerating substance to be freely movable, muchsmoother contact with the blood is enabled, and the efficiency of theblood activation can be improved.

In the aforementioned blood component separator, the specific gravity ofthe blood coagulation accelerating substance may be greater than theaforementioned each blood component separated from the blood. Hence, theprepared serum can be readily removed from the blood reservoir.

Moreover, when a serum is prepared from the blood, centrifugalseparation should be carried out after the activation of the factors tobe activated such as platelets and blood coagulation factors. However,in order to reduce damage to erythrocytes (hemolysis) and breakage ofthe blood reservoir in this case, appearing shape of the bloodcoagulation accelerating substance is preferably formed to be nearlyspherical. Furthermore, with the aim of rapid activation, the surface ofthe blood coagulation accelerator is preferably formed with a layercomprising a silicon dioxide compound.

Examples of the silicon dioxide compound which may be used include atleast one or more selected from glass, silica, diatomaceous earth,kaolin and the like, but this is not limited thereto.

Moreover, when a magnet is used as a core of the blood coagulationaccelerator, stirring of the blood can be effected by allowing amagnetic field to act on the blood reservoir, thereby permitting thefactors to be activated to be rapidly activated.

The blood coagulation accelerating substance preferably has a porousstructure in order to execute activation, because great surface area perunit volume can be provided. However, in this instance, it is necessaryto ensure penetration of blood into the pores.

In connection with the blood coagulation accelerating substance in theblood reservoir, to define the surface area to satisfy the relationshipof from 0.1 mm²/ml to 25 mm²/ml in the volume of the blood which can bereserved in the blood reservoir is preferred in light of both terms ofpromotion of activation and suppression of hemolysis. This definitioncorresponds to the best condition value at present; however, valuesoutside this range are within the scope of the present invention as longas similar effects are achieved.

In the aforementioned blood component separator, the blood reservoir mayor may not be charged with a serum separating agent.

Also, in the blood component separator, at least two connection portsare formed at the blood reservoir. Among the two connection ports, onemay be connected in an air-tight manner to an introducing path forintroducing the blood to the blood reservoir, while another may beconnected in an air-tight manner to a discharging path for dischargingeach blood component separated from the blood in the blood reservoir.

According to this invention, connection of the introducing path anddischarging path to the connection ports formed at the blood reservoircan protect the serum from contamination with blood components.

In the aforementioned blood component separator, the component storagepart comprises two or more bags. The discharging path is constitutedwith a plurality of discharge tubes connected to each bag. At least apart of the plurality of discharge tubes may be used in combination.

According to this invention, due to the component storage partcomprising two or more bags, and at least a part of the plurality ofdischarge tubes being used in combination, respective bags are connectedtogether, thereby facilitating handling. In addition, by air tightconnection of each part in this manner, contamination by microorganismscan be prevented.

In the aforementioned blood component separator, the blood reservoir maycontain air. The air contained in the blood reservoir is preferably from0.03 cc/ml to 1 cc/ml per the volume of the reservable blood.

According to this invention, inclusion of the air in the blood reservoirenables achieving an effect that is similar to the case in which theblood coagulation accelerating substance is included.

Furthermore, the method of separating blood of the present invention ischaracterized in that the blood is separated into humoral components andnonhumoral components in the presence of the humoral components and theplatelets derived from the blood using the blood component separator towhich a serum producing function which produces a serum in a practicallyusable amount is imparted by a coagulation activating action.

Specifically, the following method is provided.

A method of separating blood in which an apparatus for separatingcollected blood into a plurality of blood components and storing them isused, the apparatus being a blood component separator comprising a bloodreservoir for holding the blood to which a serum producing function thatproduces a serum in a practically usable amount as a serum is imparted,and the method comprising a reservation step of reserving the collectedblood in the blood reservoir, an activation promoting step of initiatingthe serum producing function and promoting activation of factors to beactivated including platelets and coagulation factors in the bloodreserved in the blood reservoir, and a separation step of separating thefactors to be activated from the blood which were activated andagglutinated in the activation promoting step.

In the above method of separating blood, the activation promoting stepmay be a step of shaking the blood component separator.

Furthermore, in the above method of separating blood, the serumproducing function may be provided by a blood coagulation acceleratingsubstance provided inside of the blood reservoir.

Moreover, in the above method of separating blood, each blood componentdischarged in the discharge step may include a serum.

Additionally, in the method of separating blood, the discharging stepmay be a step of storing the blood components in the component storagepart by letting the liquid components in each blood flow in the state ofthe blood coagulation accelerating substance fixed.

Furthermore, in the above method of separating blood, the bloodcoagulation accelerating substance in the discharging step may be fixedby a fixing device mounted on at least one of the interior or theexterior of the blood reservoir.

Furthermore, in the method of separating blood, the fixing device may beone or more selected from the group consisting of magnets, clamps,holding parts that hold the blood reservoir, and a plurality ofprotrusions disposed in the blood reservoir.

According to such an invention, collection of a large amount of blood ata time and quick separation are made possible. Moreover, because theprocess from the reservation step to the introduction step can beconducted without exposing to the outside air, risk of contamination bymicroorganisms can be reduced. Furthermore, the method may comprise adischarging step of discharging each blood component, other than thefactor to be activated separated from the blood in the separation step,to the component storage part.

In the activation promoting step, the factor to be activated can beactivated by shaking the entirety of the blood component separator orthe blood reservoir. Although the method of the shaking is notparticularly limited as long as hemolysis is not caused, it ispreferably conducted at a speed of shaking which allows the bloodcoagulation accelerating substance to evenly move in the blood. Also, inthe case in which a blood coagulation accelerating substance including amagnet used as a core is used, stirring of the blood can be performed byallowing a magnetic field to act on the blood reservoir.

In the separation step, fibrin is generated in the state of theactivated factor to be activated being adhered to the blood coagulationaccelerating substance. Accordingly, recovery of the serum isfacilitated. Also, in the discharging step, recovery of erythrocytes isfurther facilitated by fixing a blood coagulation acceleratingsubstance, to which fibrin was adhered, by a fixing device.

Furthermore, the present invention provides the following method ofrecovering fibers in blood and regenerative medical process.

A method comprises: separating blood into humoral components andnonhumoral components using a blood component separator to which a serumproducing function which produces a serum in a practically usable amountin the presence of humoral components and platelets derived from bloodis imparted by a coagulation activating action; and recovering fibersfrom the nonhumoral components.

A regenerative medical process comprises: separating blood into humoralcomponents and nonhumoral components using a blood component separatorto which a serum producing function which produces a serum in apractically usable amount in the presence of humoral components andplatelets derived from the blood is imparted by a coagulation activatingaction; adding the humoral components to a medium; culturing byinoculating cells collected from a subject to this medium; and preparingthus resulting cell or tissue for transplantation to the subject.

In the aforementioned regenerative medical process, the humoralcomponents may include a serum.

A regenerative medical process comprises: separating blood into humoralcomponents and nonhumoral components using a blood component separatorto which a serum producing function which produces a serum in apractically usable amount in the presence of humoral components andplatelets derived from the blood is provided by a coagulation activatingaction; adjusting the concentration of the nonhumoral components; andthereafter preparing for blood transfusion to the subject.

In the method of separating blood, the nonhumoral components may containa serum, leukocytes and erythrocytes.

Use of these methods enables preparation of a large amount of serum, andtherefore, preparation of autoserum in an amount required for cellculture is permitted. Furthermore, also in the case in which bloodtransfusion is required in surgery, because erythrocytes and fibrin canbe recovered after collecting the serum, autotransfusion after adjustingthe concentration, use of the fibrin as a scaffold of stem cells or abarrier of wounds and the like can be performed.

As explained in the foregoings the blood component separator of thepresent invention is used for separating the collected blood into eachcomponent. Because it has a blood reservoir for holding the blood, and aserum producing function is provided to this blood reservoir, a serum inwhich propagation of microorganisms is suppressed can be preparedquickly and in large amounts. Thus, it is suited for preparation oflarge amounts of serum which may be used in stem cell culture inregenerative medicine. Additionally, erythrocytes and other componentsafter collecting the serum are suited for use as the blood forautotransfusion and as a barrier for wounds.

Therefore, when the blood component separator according to the presentinvention is used, large amounts of blood components including serum canbe prepared (produced) quickly and efficiently from the collected bloodwhile ensuring high safety.

Also, in the method of separating blood of the present invention, theblood component separator according to the present invention describedabove is used, the method comprises: a holding step of holding thecollected blood in the blood reservoir; an activation promoting step ofinitiating the serum producing function and promoting activation offactors to be activated including platelets and coagulation factors inthe blood held in the blood reservoir; and a separation step ofseparating from the blood the factors to be activated which wereactivated and agglutinated in the activation promoting step, and furthercomprises, as needed, recovering the blood components remaining afterthe serum collection, and preparing for use in autologous bloodtransfusion and as a scaffold of stem cells or a barrier for wounds.

Therefore, when this method of preparing a serum is used, bloodcomponents which are highly safety can be produced in large quantities.

Also, the regenerative medical process of the present inventioncomprises: adding the serum prepared using the method of separatingblood described above to a medium; culturing by inoculating stem cellscollected from a subject to this medium; and preparing the cells or thetissue obtained by the culture for transplantation to the subject oftherapy. In addition, when the subject is a patient with low volume ofcirculating blood, such as a child, or when heavy bleeding intransplantation is feared, the recovered erythrocytes may be subjectedto transfusion, or fibrin may be used as a scaffold of the transplantedsite or as a barrier of the transplanted site.

Accordingly, use of this regenerative medical process enables use of alarge amount of serum with biological safety ensured. Hence, tissues andfunctions of the subject can be regenerated safely and certainly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view illustrating a blood component separator according to afirst Embodiment of the present invention.

FIG. 2 is a view illustrating a procedure of from collection of theblood to preservation of a serum by the blood component separatoraccording to the first Embodiment of the present invention.

FIG. 3 is a view illustrating a state of shaking a blood reservoir 10 ofthe blood component separator according to the first Embodiment of thepresent invention.

FIG. 4 is a view illustrating a state of melting of a tube 41 of theblood reservoir 10 of the blood component separator according to thefirst Embodiment of the present invention.

FIG. 5 is a cross sectional view illustrating the blood reservoir 10after centrifugal separation with the blood component separatoraccording to the first Embodiment of the present invention.

FIG. 6 is a cross sectional view illustrating a method of discharging aserum 71 prepared in the blood reservoir 10 into a bag for preservingserum 21 of the blood component separator according to the firstEmbodiment of the present invention.

FIG. 7 is a view illustrating a state of melting of a tube 42 of the bagand the blood reservoir 10 of the blood component separator according tothe first Embodiment of the present invention.

FIG. 8 is a view illustrating a bag 21 following the melting step in theblood component separator according to the first Embodiment of thepresent invention.

FIG. 9 is a perspective view illustrating the blood reservoir 10 in thedischarging step following centrifugal separation of the blood componentseparator according to the first Embodiment of the present invention.

FIG. 10 is a perspective view (partial cross sectional view)illustrating a coagulant adhered glass processed body 14 according tothe blood component separator according to a second Embodiment of thepresent invention.

FIG. 11 is a view illustrating a blood component separator 1 accordingto a fourth Embodiment of the present invention.

FIG. 12 is a view illustrating a blood component separator 1 accordingto a fifth Embodiment of the present invention.

FIG. 13 is a view illustrating a blood component separator 1 accordingto a sixth Embodiment of the present invention.

FIG. 14 is a view illustrating a blood component separator 1 accordingto a seventh Embodiment of the present invention.

FIG. 15 is a view illustrating a step of introducing physiologicalsaline solution into a blood reservoir 10 of the blood componentseparator 1 according to the fifth Embodiment of the present invention.

FIG. 16 is a characteristic view illustrating a relationship betweentime elapsed following collection of the blood and residual ratio of theplatelets according to Example 1.

FIG. 17 is a characteristic view illustrating a relationship between thearea of the glass processed body in contact with the blood and amount ofrelease of a cell growth factor (TGF-β1) into the serum according toExample 2.

FIG. 18 is a characteristic view illustrating a relationship between thearea of the glass processed body in contact with the blood and amount ofrelease of a cell growth factor (PDGF-BB) into the serum according toExample 2.

FIG. 19 is a characteristic view illustrating a relationship between thearea of the glass processed body in contact with the blood and amount ofrelease of hemoglobin into the supernatant according to Example 3.

FIG. 20 is a view illustrating a relationship between each sample andcell number according to Example 4.

FIG. 21 is a view illustrating time dependent alteration of recoveryrate of erythrocytes of each sample according to Example 5.

FIG. 22 is a view illustrating ratio of release of a growth factor andcoagulation time of platelet-rich plasma of each specimen according toExample 6.

FIG. 23 is a view illustrating residual ratio of platelets in eachspecimen according to Example 7.

FIG. 24 is a functional block diagram illustrating a constitution of theserum preparation apparatus according to an eighth Embodiment.

FIG. 25 is a schematic view illustrating a constitution of a flexiblebag according to a ninth Embodiment.

FIG. 26 is a functional block diagram illustrating a constitution of theserum preparation apparatus according to a tenth Embodiment.

FIG. 27 is a schematic view illustrating a constitution of the serumpreparation apparatus according to a twelfth Embodiment.

FIG. 28 is a schematic view illustrating a constitution of the serumpreparation apparatus according to a thirteenth Embodiment.

FIG. 29 is a schematic view illustrating a constitution of the serumpreparation apparatus according to a fourteenth Embodiment.

FIG. 30 is a schematic view illustrating a constitution of the serumpreparation apparatus according to a fifteenth Embodiment.

FIG. 31 is a schematic view illustrating a constitution of the serumpreparation apparatus according to a sixteenth Embodiment.

FIG. 32 is a schematic view illustrating a constitution of the serumpreparation apparatus according to a seventeenth Embodiment.

FIG. 33 is a schematic view illustrating a constitution of the serumpreparation apparatus according to a nineteenth Embodiment.

FIG. 34 is a schematic view illustrating a constitution of the serumpreparation apparatus according to the nineteenth Embodiment.

FIG. 35 is a schematic view illustrating a constitution of the serumpreparation apparatus according to the nineteenth Embodiment.

BEST MODE FOR CARRYING OUT THE INVENTION

The blood component separator according to the present invention is anapparatus which can produce a serum in a practically usable amount byproviding a blood coagulation accelerating function to a place forreserving blood components to produce the serum.

In a suggested typical example of the mode of the apparatus, at leastone or more flexible bags are connected aseptically in a liquid-tightand air-tight manner, to which a blood coagulation accelerating functionis provided.

Such a blood component distribution and storage apparatus has a form inappearance that is similar to blood bag or separation bag generallyreferred to. Such a form that is similar to these was adopted because ofperformances exhibited for many years to demonstrate that these aresuited forms for distributing collected various types of componentsaseptically in a liquid-tight and air-tight manner.

Additionally, the aspect of imparting a blood coagulation acceleratingfunction relates to an essential feature of the present invention. Evenin the case of a mere flexible bag, a serum can be produced throughactivation of blood coagulation factors over time when humoralcomponents including the coagulation factors are stored. However, inaddition to the necessity of a relatively long time, growth factorsincluded in the serum have come to be inactivated, and thus resultingactivity is insufficient. Hence, the blood coagulation acceleratingfunction is provided. Accordingly, humoral components including theblood coagulation factors and the blood coagulation factors are broughtinto contact in the above place, thereby accelerating blood coagulationrapidly. As a result, preparation of the serum can be carried out in anextremely short period of time, and the serum can be prepared in a statewith suppression of decreased activity. This event is applicable also tothe cases in which the bag itself is formed with a material having ablood coagulation accelerating function. Therefore, such modes are notexcluded from the present invention. In other words, a principle of thepresent invention also involves modes having a reservoir space wallformed with a material having a high blood coagulation acceleratingfunction in comparison with medical materials generally used for bloodbags, separation bags, and the like.

Examples of the substance having a blood coagulation acceleratingfunction which has been verified to have the highest effect at presentinclude glass processed bodies. It is conventionally known that glasseshave a blood coagulating action; however, there is not yet any exampleof positive use thereof for producing a relatively large amount ofserum. The present inventors tried to use a glass processed body as asubstance having a blood coagulation accelerating function to allow itto exist in blood components that produce a serum, and repeated mutualcontact. Consequently, a large amount of serum was successfully producedin a short period of time without decreasing the activity.

Additionally, as a substance having a blood coagulation acceleratingfunction, air was used in place of the aforementioned glass processedbody. It is already known that blood is coagulated upon contact with theair; however, no example is known in which the air is positively usedfor producing a serum. Thus, attempts to bring the blood into contactwith the air as a substance having a blood coagulation acceleratingfunction were repeated, and also, a large amount of serum wassuccessfully produced in a short period of time.

It has been revealed that the substance having a blood coagulationaccelerating function is activated to a greater extent as the contactwith the blood is increased. More specifically, it has been revealedthat in the case of the glass processed body, to give greater specificsurface area will be effective, while in case of the air, giving greatercontent will be effective. However, when a substance is used as asubstance having a blood coagulation accelerating function such as aglass processed body, fears of hemolysis must be considered.

Next, the following mode is suggested as a mode to bring humoralcomponents containing blood coagulation factors into contact with asubstance having a blood coagulation accelerating function (hereinafter,referred to as “blood coagulation accelerating substance”).

In the first mode, the blood collected from a human body is reserved ina blood reservoir holding a blood coagulation accelerating substance topromote coagulation of the blood, thereby separating a serum.

In this operation, the separated serum is transferred aseptically to abag other than the blood reservoir to be preservable in this bag.Alternatively, the separated serum may be aseptically discharged outsideto a subject for use as an addition to a cell culture system as a growthfactor. Also, other blood components separated concomitantly withseparation of the serum (erythrocytes, fibrin, and the like) can be usedfor blood transfusion or regeneration therapy.

Second, a mode is suggested in which the blood collected from a humanbody is reserved in a blood reservoir, and after separating bloodcomponents in the state of this reservoir including anticoagulant addedthereto, platelets and humoral components including blood coagulationfactors which are components that produce a serum, among the separatedcomponents, are aseptically transferred into separate bags, and a serummay be produced in this bag. In this instance, although theaforementioned blood coagulation accelerating substance should be storedin the bag for producing a serum, as a matter of course, function of thestored coagulation accelerating substance can be sufficiently fulfilledby adding a neutralizing agent for neutralizing the contaminatedanticoagulant. In this procedure, the separated serum may be preservedin the blood reservoir without modification, or may be asepticallytransferred to and preserved in another bag. The separated serum may beaseptically discharged outside to uses such as addition to a cellculture system as a growth factor. Also, other blood componentsseparated before and after separation of the serum (erythrocytes, fibrinand the like) can be used for blood transfusion or regeneration therapy.

The third mode has a relationship with the second mode. In this mode,the serum is produced in a similar manner to the second mode aftercollecting only the components that produce the serum from the bloodbefore the introduction to the aforementioned apparatus. In this mode,so called apheresis is carried out in the first place, and therefore,unnecessary components can be immediately returned to the subject incase in which collection of the serum or fibrin alone is intended.Accordingly, a physical burden imposed on the subject can be diminished.

Hereinafter, the present invention will be explained in more detail.

First Embodiment

Overall Constitution of Blood Component Separator 1

Constitution of a blood component separator 1 according to thisEmbodiment will be explained with reference to FIG. 1. In FIG. 1, only aprincipal part in the constitution of the blood component separator 1 isextracted and illustrated.

As shown in FIG. 1, the blood component separator 1 according to thisEmbodiment is constituted from a blood reservoir 10 and a componentstorage part 20 as main elements. Among these, the blood reservoir 10 isconstituted from a main body part 11 formed with two sheets of aflexible resin material, for example, soft polyvinylchloride, by fusionat the external marginal part 11 a to yield a bag shape, and a glassprocessed body 12 disposed inside of the main body part 11. In otherwords, the blood reservoir 10 has a constitution including glassprocessed bodies 12 as a blood coagulation accelerating substance storedinside of the main body part 11 as an exterior package.

The blood reservoir 10 is similar to so-called blood bag and transferbag in respect of inner volume and shape in the main body part 11;however, it is different from the blood bag in terms of absence of ananticoagulant filled therein, and also different from transfer bag interms of inclusion of a coagulation accelerating agent. In addition, theinside of the blood reservoir 10 is previously subjected to asterilization.

The glass processed bodies 12 in the main body part 11 have asubstantially spherical shape and consists of, for example, soda glass.Also, FIG. 1 shows a constitution having three glass processed bodies 12in the blood reservoir 10; however, it is preferred to define thesurface area of the glass processed body 12 to satisfy a relationship tothe volume of reservable blood to be 0.1 mm²/ml or greater, in terms ofachieving a blood coagulation accelerating function.

The glass processed bodies 12 are not joined to the inner wall of themain body part 11 or the like, but are provided to give a freely movablestate in the main body part 11 when a shaking action, a vibratingaction, or the like is applied to the main body part 11.

Moreover, in order to suppress breakdown of erythrocytes in the bloodwhich may lead to hemolysis when a shaking action, a vibrating action orthe like is applied to the main body part 11 after holding the blood,and furthermore, when the blood is subjected to a centrifugal separator,it is preferred to provide the glass processed body 12 with the surfacearea to satisfy a relationship of the surface area thereof to the volumeof reservable blood in the main body part 11 of the blood reservoir 10to be 25.0 mm²/ml or less. Grounds of the preference of these rangeswill be described later.

The component storage part 20 is constituted from 6 bags 21 to 26, eachbeing made with soft polyvinylchloride. These are previously subjectedto a sterilization.

As shown in FIG. 1, two tubes 41 and 42 are connected in an air-tightmanner at the upper edge end of the main body part 11 of the bloodreservoir 10 to the connection ports thereof, respectively. The tube 41among them plays a role as an introducing path for introducing theblood, and therefore, a needle for collecting blood 30 or a junctionwhich can be connected to a needle for collecting blood is connected atthe other end. Another tube 42 connected in an air-tight manner to theblood reservoir 10 is connected to each of the bags 21 to 26 via tubes43 to 46 and 51 to 56, and branches 61 to 65. These play a role asdischarging path for discharging separated blood components. These tubes41 to 46 and 51 to 56 are constituted from a resin material havingflexibility, for example, a material such as soft polyvinylchloride orthe like. In this constitution, the bags 21 to 26 and each tube 51 to 56of the component storage part 20 are also connected in an air-tightmanner.

Additionally, regarding the dimensions of the tubes 41 and 42, the innerdiameter size is defined to be smaller than the external diameter sizeof the glass processed body 12. This prevents the glass processed body12 from gaining entry into the tubes 41 and 42 when the serum isprepared and discharged.

The blood reservoir 10, and the bags 21 to 26 and each of the tubes 41and 42, and 51 to 56, are connected in a state to make the inner spaceinaccessible to the external environment. Also, each of the tubes 42 to46 and 51 to 56 and each of the branches 61 to 65 are connected instates to make the inner region where the serum circulates inaccessibleto the external environment. Specifically, they are connected by solventadhesion, thermal welding, ultrasound welding or the like.

Although not shown in FIG. 1, the blood component separator 1 accordingto this Embodiment is constituted to be able to switch the flow channelwhen the blood and the extracted serum are discharged through pinchingthe necessary site of each tube 42 to 46, and 51 to 56, with a clamp.

Operation for Serum Preparation

Operation for serum preparation using the blood component separator 1having the constitution as described above will be explained withreference to FIG. 2 to FIG. 8. FIG. 2 will be referred to freely incombination for explaining the operation.

As shown in FIG. 2, operation for separating blood using theaforementioned blood component separator 1 is constituted from sevensteps (S1 to S7) as generally classified.

First, in the first step of the operation, the needle for collectingblood 30 shown in FIG. 1 is stuck into patients, and blood is collected.In this step, the blood collected through the needle for collectingblood 30 is held in the blood reservoir 10 positioned down below via thetube 41 (reservation step S1). A breakable partition wall is mountedbetween the tube 42 and the blood reservoir 10 such that the collectedblood in the blood reservoir 10 does not flow into the component storagepart 20. Alternatively, the channel of the tube 42 is closed at the footside of the blood reservoir 10 using a clamp or the like. Thereservation step S1 is terminated after collecting a required amount,taking into account the patient's physical condition upon collecting theblood. The required amount referred to herein may be approximately 200to 600 ml when there is no problem in the physical constitution andphysical condition of the patient.

Upon collecting the blood, a blood collecting device which has beenextensively used in blood donation or the like may be also used.

Next, as shown in FIG. 2, after initiating the reservation step S1, theblood reservoir 10 is shaken in parallel therewith (activation promotingstep S2). As shown in FIG. 3, the blood reservoir 10 reserving thecollected blood is gently agitated by a shaking apparatus 100 to bebrought into contact with the glass processed bodies 12 stored inside.Then, the platelets and coagulation factors included in the blood arecoagulated on the surface of the glass processed body 12, and from theplatelets activated during the coagulation are released growth factorsderived therefrom. (Also, this activation promoting step carried out ata low temperature is effective in acceleration of plateletagglutination.)

Because the blood reservoir 10 is formed to have the external size thatis equivalent to common blood bags, any well known shaking apparatus canbe used for shaking the blood reservoir 10. Additionally, although notshown in FIG. 3, the blood reservoir 10 is connected to each of the bags21 to 26 via each of the tubes 41 to 46 and 51 to 56, and therefore,shaking can be also executed after folding at these connections.

Following the reservation step S1, the needle for collecting blood 30 isdrawn to remove from the subject of collection of the blood, and then apart of the tube 41 connecting the needle for collecting blood 30 andthe blood reservoir 10 is subjected to melting, and welding of itsmelting edge (melting step S3) is perfected at the same time. Formelting of the tube 41, a melting machine 110 as shown in FIG. 4(generally referred to as a sealer) can be used to complete melting.

On the other hand, the blood reservoir 10 separated from the patientproceeds through the activation promoting step S2 together with thecomponent storage part 20 and each of the tubes 42 to 46 and 51 to 56connecting therebetween, as well as branches 61 to 65 and the like. Theyare taken together to be compact, and are subjected to a centrifugeseparation (centrifugal separation step S4). The tube 42 then is kept inthe state with the channel being closed by a breakable partition wall ora clamp, similarly to the case of the reservation step S1.

When the blood is collected through adding an anticoagulant previously,the melting step S3 and the centrifugal separation step S4 may becarried out prior to the activation promoting step S2. In this instance,the centrifugal separation may be conducted under the followingconditions:

centrifugal separation of whole blood: 4,400 g×4 to 6 min, 2,250 g×10min; and

centrifugal separation of platelet-rich plasma (PRP): 1,100 g×4 to 6min.

Conditions for centrifugal separation of the blood reservoir 10 may bedefined depending on the amount of the reserved blood and type of thecomponents to be separated, however, for example, they may be defined tobe, e.g., 2250 g×10 min, at 4° C. With respect to the blood reservoir 10following the centrifugal separation, explanation will be made withreference to FIG. 5.

As shown in FIG. 5, in the blood reservoir 10 subjected to thecentrifugal separation after proceeding through the activation promotingstep S2, the blood is generally separated into three layers of serum 71,leukocytes 72 and erythrocytes 73, although they may vary depending onthe centrifugal separation conditions. Additionally, glass processedbodies 12 are lying down in the state with a coagulant 74 of theplatelets and coagulation factors adhered on the surface thereof(hereinafter, referred to as “coagulant adhered glass processed body14”) at the bottom of the main body part 11 a in the blood reservoir 10.This coagulant adhered glass processed body 14 includes, as shown in theenlarged portion of the Figure, the coagulant 74, which was formed bycoagulation of the platelets and coagulation factors, adhered on thesurface of the glass processed body 12. In order to discriminate thepresence or absence of adherence of the coagulant herein, a referencenumeral 14 is assigned to the glass processed body after the adherenceof the coagulant 74.

As described above, the serum 71 that is a supernatant component in thestate shown in FIG. 5 comprises growth factors derived from theplatelets and coagulation factors sufficiently released therefrom in theactivation promoting step S2. Also in the centrifugal separation stepS4, because both of the two tubes 41 and 42 connected to the upper edgeend of the main body part 11 of the blood reservoir 10 in an air-tightmanner are kept in a closed state to be inaccessible to the externalenvironment, invasion of microorganisms and the like is prevented.

Referring back to FIG. 2, the factor to be activated, which wasactivated in the activation promoting step S2 through the centrifugalseparation step S4, forms a clot and is separated from the blood(separation step S5).

Furthermore, in the separation step S5, the serum 71 separated andextracted in the blood reservoir 10 is sequentially divided into all ora part of the bags 21 to 26 in the component storage part 20(discharging step S6). A method for discharging will be explained withreference to FIG. 6.

As shown in FIG. 6, when discharge of the extracted serum 71 into thebag 21 of the component storage part 20 is intended, channel of the tube43 is closed using a clamp 90, and the blood reservoir 10 is compressed(F1) in this state with a pressurizer 80 placed outside of the bloodreservoir 10. As described above, the tube 41 connected to the bloodreservoir 10 in an air-tight manner is subjected to melting at itsmidstream 41 a when the reservation step S1 is completed, and at thesame time, its end and the vicinity 41 b are welded.

Hence, a part of the serum 71 that is the supernatant part extracted bythe separation is discharged into the bag for preserving serum 21 viathe tube 42, the branch 61 and the tube 51 through receiving thecompression F1.

Closure of the channel of the tube 43 can be performed by pinching theflexible tube 43 between a circular disc 91 and a base 92 of the clamp90.

Referring back to FIG. 2, after packing the bag 21 with the serum 71 ina required amount, the tube 51 is subjected to melting and welding(melting step S7). This melting and welding may be performed using amethod that is similar to melting and welding of the tube 42 prior tothe aforementioned centrifugal separation step S4, as shown in FIG. 7.Moreover, as shown in FIG. 8, the bag 21 including the serum 71 packedtherein is subjected to a preservation treatment such as, e.g., freezingpreservation.

This discharging step S6 and melting step S7 is sequentially carried outon each of the bags 21 to 26, and the operation for serum preparation isterminated when the serum 71 is packed in all or a part of the bags 21to 26. Additionally, as needed, the erythrocytes 73 may be washed anddiluted with an anticoagulant such as physiological saline solution,CPD, or an ACD-A solution, or solution for preserving blood such as MAP,and can be preserved as blood for transfusion. This method will bedescribed later.

Significance of Blood Component Separator 1

In the blood component separator 1 according to this Embodiment, theblood reservoir 10 is constituted from the main body part 11 having aninternal volume that is equivalent to the blood bags, and the glassprocessed bodies 12 are provided inside thereof. Because the serum isprepared in this blood reservoir 10 from the blood, a larger amount ofserum can be prepared at a time in comparison with cases in which aconventional blood collection tube is used, thereby accomplishingadvantages in light of steps in the preparative operation, and the like.Furthermore, this may reduce risk of contamination of the prepared serumwith microorganisms and the like, and therefore, the blood componentseparator 1 is also suited for preparing a serum having high safety.

Additionally, because the glass processed bodies 12 are stored as ablood coagulation accelerating substance in the blood componentseparator 1, adherence of the clot onto the surface of the glassprocessed body 12 in preparation of the serum, and contamination withfibrin and clot in the serum during fractionation of the serum can beprevented.

Moreover, in the blood component separator 1, in addition to theaforementioned advantages, the blood or the serum is not exposed to theexternal environment because the blood reservoir 10, and the bags 21 to26 and each of the tubes 41, 42, and 51 to 56 are connected in anair-tight manner to make the inner space inaccessible to the externalenvironment. Thus, even higher safety is ensured.

Furthermore, because the aforementioned tubes 41, 42, and 51 to 56 haveflexibility, and are formed with a material that permits melting andwelding, the inside thereof is not exposed to the external environmentwhen these channels are cut off in a timely manner. Therefore, also inthis respect, the blood component separator 1 can be referred to ashaving high safety.

In general, when blood is held in a container not including anyanticoagulant (for example, ACD-A liquid or the like), platelets andcoagulation factors in the blood gradually coagulate after leaving thecontainer to stand for 20 minutes or longer after collection of theblood. However, when the amount of the air in the container is small, inparticular, coagulation of the blood is not accelerated, thereby causingthe problem of requirement of a long period of time until coagulation isaccomplished.

In contrast, in the blood component separator 1 according to thisEmbodiment, because glass processed bodies 12 as a blood coagulationaccelerating substance having a blood coagulation accelerating functionare disposed inside the blood reservoir 10 for holding the blood, mostplatelets (for example, 95% or more of the platelets) and coagulationfactors are caused to coagulate within 10 minutes, thereby enablingoperation of the serum preparation to be quickly performed. Accordingly,when the blood component separator 1 according to this Embodiment isused, the serum can be quickly prepared. Furthermore, becauseerythrocytes remaining in the blood component separator 1 afterpreparing the serum via the aforementioned activation promoting step S2scarcely form clot, there is a possibility of reuse of the erythrocytesas a component for blood transfusion.

Moreover, in the field of regenerative medicine, for example, serumshaving high safety are required for culturing stem cells. A large amountof serum can be prepared and preserved in a safe and quick manner bypreparation and preservation of the serum 71 using the blood componentseparator 1 as described above. Accordingly, when the blood componentseparator 1 according to this Embodiment is used, regeneration therapyfor a tissue or a function can be performed with high efficiency on apatient while securing high safety.

Also, in the above Embodiment, activation of platelets and coagulationfactors in the blood can be further promoted when the glass processedbody 12 was defined to have a surface area so as to satisfy arelationship of from 0.1 mm²/ml or greater in the volume of the bloodwhich can be reserved in the blood reservoir 10. Moreover, when thesurface area of the glass processed body 12 in the volume of reservableblood is defined to fall within the range of from 0.1 mm²/ml to 25mm²/ml, simultaneous achievement of both suppression of hemolysis in theactivation promoting step S2 and the centrifugal separation step S4, andpromotion of activation of the platelets and coagulation factors may beenabled.

Number of the glass processed bodies 12 in the blood reservoir 10 wasspecified to be three in this Embodiment, but this is not limitedthereto. The number of stored glass processed bodies 12 may be from 1 to50 from a practical point of view.

Moreover, although glass processed bodies 12 were used as a bloodcoagulation accelerating substance in the blood component separator 1,the blood coagulation accelerating substance is not limited thereto. Forexample, any one that constitutes the contact region with the blood withan inorganic substance consisting of at least one selected from silicondioxide compounds such as silica, diatomaceous earth and kaolin may bealso used. Furthermore, the substance provided at the contact region isnot limited to an inorganic substance.

Additionally, the blood coagulation accelerating function was impartedto the blood reservoir 10 by storing the blood coagulation acceleratingsubstance (glass processed body 12) having a blood coagulationaccelerating function in the blood reservoir 10 according to theaforementioned blood component separator 1; however, any constitution ispermitted as long as the blood coagulation accelerating function isachieved even though a blood coagulation accelerating substance is notnecessarily stored in the blood reservoir 10. For example, a part of theinside wall in the main body part 11 of the blood reservoir 10 may becovered by the aforementioned inorganic substance or the like.

Furthermore, the shape of the glass processed body 12 in the bloodcomponent separator 1 was made substantially spherical; however, theshape of the blood coagulation accelerating substance according to thepresent invention is not limited thereto. However, with respect toreduction of occurrence of hemolysis and breakage of the bag and thelike upon centrifugal separation or the like, the outer surface (contactregion with the blood) is preferably formed to give a continuous curvedface.

In the discharging step S6, as shown in FIG. 9, the erythrocytes can bedischarged through blocking the glass processed bodies, to which fibrinwas adhered, with a holding part 16 such as, for example, a clamp fromthe outside of the blood reservoir 10 to separate the glass processedbodies including the platelets and coagulation factors adhered onto thesurface thereof. This holding part 16 preferably has a face with a waveform or an uneven shape to be in contact with the blood reservoir 10 sothat washing fluids of erythrocytes and fibrin and the like can bepassed through even after pinching the blood reservoir 10.

Furthermore, although solid glass processed body 12 was used in theEmbodiment of the present invention, it may not be necessarily solid aslong as a substance having a blood coagulation accelerating function isformed on its outer surface. For example, it may have a porousstructure, and the entire region to be in contact with the bloodincluding the wall face in the pores may be coated with a silicondioxide compound such as glass.

Second Embodiment

The blood component separator according to this Embodiment has adifference with the aforementioned blood component separator 1 accordingto the first Embodiment, in the mode of the glass processed body storedin the blood reservoir 10. Thus, the mode of the glass processed bodywill be explained below with reference to FIG. 10, although descriptionof other parts will be omitted.

As shown in FIG. 10, the coagulant adhered glass processed body 14according to this Embodiment is similar to the glass processed body 12described above in terms of having a shape which is substantiallyspherical; however, it is characterized in having a two-layeredstructure including a core body part 141 and a superficial part 142.

Among the two layers constituting the coagulant adhered glass processedbody 14, the core body part 141 is constituted with a magnet. On theother hand, the superficial part 142 is constituted with, for example,soda glass, as an inorganic substance that is identical to the materialconstituting the aforementioned glass processed body 12.

When the blood reservoir 10 storing therein the coagulant adhered glassprocessed bodies 14 having such a structure is used, an effect tofacilitate stirring of the blood can be achieved by allowing a magneticfield to act on the blood reservoir 10 while shaking in the activationpromoting step S2 shown in FIG. 2. More specifically, by allowing amagnetic field to act on the blood reservoir 10 from outside with, forexample, a stirrer such as a magnetic stirrer, rotary movements of thecoagulant adhered glass processed bodies 14 are caused in the containerto result in contact with the blood with higher efficiency.

Therefore, in the blood component separator having the coagulant adheredglass processed bodies 14 in the blood reservoir 10, activation of theplatelets and coagulation factors can be more rapidly perfected than theaforementioned blood component separator 1, thereby enabling quickerpreparation of the serum.

Additionally, when the glass processed bodies 14 are constituted with amagnet, they can be fixed with a magnet from outside of the bloodreservoir 10 in the discharging step S6.

Third Embodiment

The blood component separator according to this Embodiment has adifference with the aforementioned blood component separator 1 accordingto the first Embodiment, in that one of the bags 21 to 26 constitutingthe component storage part 20 is provided for use to let air away (seeFIG. 1). Upon collection of blood, the air is inevitably included in theblood reservoir 10 in a volume under the capacity of the tube 41;however, presence of less air is preferred in the separation step ineach blood component S5. Hence, when a bag to let air away according tothis Embodiment is provided between the blood reservoir 10 and thecomponent storage part 20, the air alone can be removed prior to theseparation step into each blood component S5. Other constitutions inthis Embodiment are similar to that in the first Embodiment, andtherefore, description of other parts is omitted.

Fourth Embodiment

The blood component separator according to this Embodiment has adifference with the aforementioned blood component separator 1 accordingto the first Embodiment, in the inclusion of the air 15 in place of theblood coagulation accelerating substance added to the blood reservoir10, as shown in FIG. 11. In this case, because there is no need topreviously add the blood coagulation accelerating substance, reductionof manufacturing cost may be accomplished. Content of the air 15 ispreferably from 0.03 cc/ml to 1 cc/ml per the volume of the reservableblood. It is preferred that the tube 41 have a mechanism for preventingleakage of the air included to give the aforementioned content, untilthe time of use.

Also, the air and the blood coagulation accelerating substance may beused in combination.

Fifth Embodiment

The blood component separator according to this Embodiment has adifference with the aforementioned blood component separator 1 accordingto the first Embodiment, in addition to the glass processed bodies 12(blood coagulation accelerating substance) to at least one of the bags21 to 26 constituting the component storage part of the blood componentseparator 1, as shown in FIG. 12. Moreover, in the case of thisEmbodiment, a citric acid neutralizing agent including calcium ions maybe further added to the container to which the glass processed bodies 12are added. In this instance, so-called “blood bag for donation” to whichan anticoagulant such as a CPD solution is added can be used as theblood reservoir 10.

The blood in the blood reservoir 10 can be separated to some extent bycentrifugal separation or the like, but the serum cannot be producedwithout modification because the anticoagulant was added. In thisEmbodiment, the blood in the blood reservoir 10 is neutralized with theneutralizing agent added to the bag 21. Thus, the growth factors in theblood are activated, thereby enabling the produced serum to bedischarged into the bag 22.

Sixth Embodiment

The blood component separator according to this Embodiment has adifference with that according to the fifth Embodiment, in the inclusionof the air 15 in place of the blood coagulation accelerating substanceadded to the bag 21 of the blood component separator 1, as shown in FIG.13. In this case, similarly to the fourth Embodiment, reduction ofmanufacturing cost may be accomplished because there is no need topreviously add the blood coagulation accelerating substance. Content ofthe air 15 is preferably from 0.03 cc/ml to 1 cc/ml per volume of theheld blood. It is preferred that the tube 51 have a mechanism forpreventing leakage of the air included to give the aforementionedcontent, until the time of use.

Also, the air and the blood coagulation accelerating substance may beused in combination.

Moreover, the blood component separator 1 according to the firstEmbodiment and the fourth Embodiment has a component storage partconstituted from six bags 21 to 26, however, number of the bagsconstituting the component storage part is not limited thereto. Inaddition, although glass processed bodies are used as the bloodcoagulation accelerating substance in the blood component separator 1according to the first Embodiment and the fourth Embodiment, similareffect may be also achieved when the air is include in place of theglass processed body.

In this instance, it is preferred to give from 0.03 cc/ml to 1 cc/ml perthe volume of the reservable blood.

Also, according to the present invention, effective utilization oferythrocytes and fibrin remaining in the blood reservoir 10 followingseparation of the blood is enabled. Hereinafter, such modes will beexplained in detail.

Seventh Embodiment

The blood component separator 1 which separated the blood in the firstEmbodiment is used. As shown in FIG. 14, the blood reservoir 10 afterseparating the blood and discharging the serum into the bag 21(component storage part) includes the coagulant adhered glass processedbodies 14 to which fibrin is adhered and residues of erythrocytes, andthe like. Physiological saline may be previously charged in any one inwhich the serum was not stored, among the component storage parts 21 to26 connected to this blood reservoir 10 in an air-tight manner,alternatively, a physiological saline-containing bag 120 which containsphysiological saline is connected to the tube 41 of this blood reservoir10 to allow for mixing with the erythrocytes in the blood reservoir 10.Accordingly, the mixture can be used as blood for transfusion (see FIG.15). Furthermore, the coagulant adhered glass processed bodies 14 towhich fibrin was adhered may be additionally washed after dischargingall blood components, and the resulting fibrin obtained after washingcan be used as a scaffold of stem cells or as a barrier for wounds.

Moreover, a constitution involving the blood reservoir and the componentstorage part being communicated with a tube therebetween was adopted asa blood component separator in the first Embodiment and the secondEmbodiment; however, the part for reserving the blood and the part forreserving the serum are not necessarily constituted as distinctcontainers. For example, one container constituted such that a partthereof can be cut away by melting, welding or the like is provided, andthe blood is held in this container, thereby providing a constitutionwhich may be used as a component storage part 20 therefrom. In addition,a clamp which pinches the blood reservoir 10 such that the glassprocessed bodies 12 to which fibrin was adhered, or the coagulantadhered glass processed bodies 14 are not discharged was demonstrated inthe discharging step S6, but is not limited thereto.

Eighth Embodiment

Execution of mechanical and automatic serum preparation and serumdivision as explained in the above Embodiments is enabled by the serumpreparation apparatus of this Embodiment. As shown in FIG. 24, thepresent serum preparation apparatus 240 is constituted from a bloodintroducing device 242 (head pressure, ejection pump or the like) foraseptically introducing the collected blood to a blood coagulationaccelerating container 241, a serum discharging device 243 (suction pumpor the like) for aseptically discharging the serum prepared by the bloodcoagulation accelerating container, a serum introducing device 245 (headpressure, ejection pump or the like) for discharging and/or distributingthe discharged serum into a plurality of serum preserving containers244, a shaking device 246 for shaking the container for preparing serumsince initiation of introduction of the blood to the container forpreparing serum, a separating device 247 for separating serum componentsby centrifugal separation of the container for preparing serum aftercompleting shaking, and a controlling device 248 for controlling eachoperation timing and operation itself. The blood coagulationaccelerating container is a container having a blood coagulatingfunction for the purpose of preparing a serum as described above, whichmay be any container having a blood coagulating function such as oneincluding a glass processed body in a flexible bag as described above,as well as a glass container or the like. Also, a container havingmixing/charging ports 511 to 516 shown in FIG. 30 may be used. In orderto maintain sterility, a structure for making the handled liquidinaccessible to the outside air may be provided so that contamination ofthe liquid with microsomes can be avoided, but the form thereof is notparticularly limited. Also, the amount of the liquid such as blood,serum or the like as described above may be monitored by a knownflowmeter, and the operation may be controlled (control of the amount ofintroduced blood, control of the amount of distribution of the serum andthe like) based on the results of the same. Moreover, followingdistribution of the serum, the serum preserving container may beautomatically sealed hermetically.

According to the aforementioned serum preparation apparatus 240, theserum is automatically prepared after collecting the blood, and theprepared serum is automatically distributed into the preservationcontainer. Therefore, preparation of the serum can be extremely easilycarried out.

When the preparation container is separately attached after preparingthe serum in the aforementioned serum preparation apparatus 240, it mayserve as a distribution apparatus so that distribution into thepreservation container can be effected.

Ninth Embodiment

FIG. 25 shows a flexible bag (container corresponding to 10 in FIG. 1)having a filter 251 for regulating the quantity of the air in a serumpreparation bag.

The filter has a function to regulate the quantity of the air throughmaking the filter open to the outside (opened by opening the cover, notshown in the Figure) after introducing the blood into the bag. Thefilter is provided to avoid the inability of recovering a bloodcomponent such as erythrocytes through generation of clot due toinsufficient performance of stirring when the quantity of the air is toogreat. Thus, a function to regulate blood coagulation velocity to be arelatively suitable value for the amount of the blood is provided byregulating the quantity of the air.

Tenth Embodiment

FIG. 26 is a functional block diagram of the serum preparation apparatushaving a function of regulating the time when the blood coagulationaccelerating container 261 is shaken for preparing the serum such thatthe amount of serum preparation is optimized to attain a sufficientlevel.

Although the constitution is similar to the apparatus shown in FIG. 24,control of shaking is executed in which the shaking time period isdefined to be a continuing predetermined time from the time point ofinitiation of introducing the blood into the container for preparingserum, up to following termination of collecting the blood. Accordingly,the serum is efficiently produced. This is an operation control whichwas realized on the basis of the inventor's empirical rule that theserum can be sufficiently produced by starting shaking from the timepoint of initiation of the collection of the blood, and shaking for atime period longer than the time period of the collection of the blood.The shaking time may be also determined on the basis of results ofmonitoring of the amount of the serum produced in effect.

Eleventh Embodiment

In this Embodiment, cases in which the serum prepared without usinghemocyte components is predominantly used are assumed. This Embodimentis characterized by the container for preparing serum in which a serumseparating agent is stored in addition to the aforementioned glassprocessed body. By including the serum separating agent in this manner,production of the serum can be facilitated through acceleration of bloodcoagulation by the glass. Thus, the produced serum can be separated fromother components well and efficiently.

Twelfth Embodiment

FIG. 27 shows a serum preparation apparatus 270 that is an apparatus forpreparing a serum in which acceleration of blood coagulation ispermitted from the collected blood, similarly to the serum preparationapparatus shown in FIG. 1. Differences between the serum preparationapparatus 270 and that shown in FIG. 1 lie in distribution of thecollected blood into a plurality of containers for preparing serum 271,preparation of the serum in the container for preparing serum 271, andtransfer and/or preservation of the prepared serum in a plurality ofpreservation containers 272. By thus distributing the collected bloodinto a plurality of containers for preparing serum 271 and preparing theserum in the container for preparing serum 271, the container forpreparing serum is more expanded, resulting in greater degree of freedomof the glass processed body. Accordingly, stirring of the glass isfacilitated to improve accessibility to the blood, thereby enablingcarrying out more efficient preparation of the serum.

Thirteenth Embodiment

FIG. 28 shows a novel serum preparation apparatus 280. The serumpreparation apparatus 280 has an indwelling needle 282 connected to acatheter 281, and a syringe 283 connected to the end of the catheter281, with the syringe 283 including a blood coagulation acceleratingsubstance 284 (glass or the like) stored therein.

According to the aforementioned serum preparation apparatus 280,preparation of the serum is enabled by introducing the collected bloodinto the syringe 283 through the catheter 281 from the indwelling needle282 while shaking the syringe 283. Thereafter, the serum can beseparated by centrifugation of the whole syringe 283. When a serum isprepared from a human body with this serum preparation apparatus 280,the operation is preferably conducted as far as possible from the humanbody while positioning the syringe 283 down below the site of collectionof the blood such that coagulation signal of the blood coagulation isnot transmitted to the human body.

Fourteenth Embodiment

FIG. 29 shows a constitution that is substantially similar to the serumpreparation apparatus shown in FIG. 1, but is greatly different in thata filter 291 is provided in a communicating path between the bag and thebag. This filter 291 is a filter having a pore with a size capable ofselectively passing the serum without passing the hemocyte components.Consequently, the serum can be conveniently separated by passing theliquid after shaking through this filter even though the centrifugalseparation step is not conducted.

Fifteenth Embodiment

FIG. 30 shows a constitution that is substantially similar to the serumpreparation apparatus shown in FIG. 1, but is different in that it is aserum preparation apparatus having each bag constituted in anaseptically connectable and detachable manner. Exemplary constitution ina connectable and detachable manner includes a constitution having ahighly air-tight mixing/charging port with a function to allowopening/closing of a slit type opening of a valve withattachment/detachment of an insertion instrument (see, Japanese PatentNo. 3389983). In this case, a luer is desirably used at the tip of thetube as a communicating path to be connected. According to such aconstitution, attachment and detachment of the bag can be readilycarried out.

Sixteenth Embodiment

FIG. 31 shows a constitution having a function that is substantiallyidentical to the blood reservoir 10 shown in FIG. 1, but it is differentin that a temporal partition 310 to be a divider is provided so as notto cause entry of the blood in the vicinity of the serum outlet duringpreparation of the serum. When the blood enters and coagulates in thevicinity of the serum outlet, recovery of the serum may be difficult.Alternatively, the coagulated blood may be contaminated in the serum tobe preserved. The temporal partition 310 is provided to be removableafter preparing the serum so that the serum can be transferred from theserum outlet to the preservation bag. Suggested temporal partition 310may be a closure by a clamp, or a closure with easy peel or the like.The temporal partition 310 may be of any mode as long as it can isolatethe outlet and the vicinity thereof from the blood during preparation ofthe serum, while the isolation can be released upon removal of theserum, if necessary.

Seventeenth Embodiment

FIG. 32 shows a constitution having a function that is substantiallyidentical to the blood reservoir 10 shown in FIG. 1, but it is differentin that a bag for flushing 320 is provided so as not to cause entry ofthe blood by the operation of preparing the serum in the vicinity of theserum outlet. When the blood enters and coagulates in the vicinity ofthe serum outlet, recovery of the serum may be difficult. Alternatively,the coagulated blood may be contaminated in the serum to be preserved.

A gas or a liquid is charged in the bag for flushing 320, and the bloodadhered to the serum outlet and in the vicinity thereof (also includingthe coagulated blood) is flushed to eliminate therefrom.

The Embodiments 16 and 17 are particularly advantageous in the case inwhich the serum preparation bag of the Embodiment 18 without having theglass processed body described below is used because the absence of theglass processed body may result in floating of the coagulated componentswhich may adhere in the vicinity of the serum outlet as the case may be,although the coagulated components adhere to the glass processed bodywhen the glass processed body is provided thereby being capable ofpreventing the adherence of the coagulated components in the vicinity ofthe serum outlet.

Eighteenth Embodiment

In this Embodiment, the glass processed body is not provided in theblood reservoir 10 shown in FIG. 1, and the serum is prepared by leavingthe collected blood to stand in the empty bag. Then, the prepared serumis transferred to the preservation bag, and is divided into a pluralityof preservation bags and is preserved.

Nineteenth Embodiment

In this Embodiment, the extensively researched aspect for preventing theglass processed body shown in FIG. 1 from entering into the tube 42 andthe like to evoke possible interference of transfer of the serum isspecifically explained.

First, the relationship between diameters of the glass processed bodyand of the tube forming the transfer path is defined so that thediameter of the glass processed body becomes greater than the internaldiameter of the tube. Accordingly, invasion of the glass processed bodyinto the tube 42 and the like can be prevented. For example, thediameter of the beads is preferably greater than the internal diameterof the tube by approximately 1 mm to 5 mm. Thus, when the internaldiameter of the tube is 3 mm, the beads preferably have a diameter ofapproximately 4 mm.

Next, as shown in FIG. 33, a plurality of spot welding parts 331 may beformed in the vicinity of the serum output tube port (formed by thermalwelding). Accordingly, transfer of the glass can be prevented. Intervalbetween spot welding parts 331 herein is smaller than the diameter ofthe beads. Furthermore, the number of the spots is desirably equal to orgreater than number of the beads, because the beads completely occludethe flow channel for removing the serum when the number of the beads issmaller than the number of the spots as shown in FIG. 35, therebyleading to unsecured flow channel for removing the serum. In contrast,when the number of spots is equal to or greater than the number of thebeads as shown in FIG. 34, the beads do not completely occlude the flowchannel for removing the serum, thereby leading to ensured flow channelfor removing the serum without fail.

Each Embodiment described in the foregoing can be perfected alone, as amatter of course, but each may be also performed in combination freely.Practice of the present invention is not limited to single Embodiment.Additionally, the prepared serum may be used to culture cells collectedfrom a patient, which cells may be transplanted into the patient, andtransfusion of the prepared hemocyte components to the patient may bealso carried out. Accordingly, they can also be used in regenerativemedicine.

Hereinafter, the present invention will be explained in more detail, butthe present invention is not in any way limited to these Examples.

Results of study on efficacy of the serum prepared using the bloodcomponent separator according to the first Embodiment, efficacy ofrecovered erythrocyte, and proliferation of stem cells will be explainedbelow. In the experiments, an exterior package made of polyvinylchloridewas used.

Example 1 Determination of Activation Promoting Effect

Large and small glass processed bodies consisting of soda glass wereadded to a blood storage part under the conditions shown in Table 1,respectively. To this blood storage part was charged 20 ml of freshhuman blood. It was incubated while stirring, and each 1.5 ml wascollected in 10, 20, 30, 60 and 90 minutes thereafter. Then, the numberof the platelets was counted. For the stirring, a stirring (shaking)apparatus (Multi Shaker MMS-300, manufactured by Tokyo Rikakikai Co.,Ltd.) was used, and for the counting of hemocytes, a hematology analyzer(Multiparameter automated hematology analyzer K-4500, manufactured bySYSMEX CORPORATION) was used.

TABLE 1 Sample A B C D E F G H I Diameter 1 mm φ 4 mm — (4 mm²/number)(50 mm²/number) Number 0 1 3 5 10 1 3 5 10 Ratio of glass surface 0 0.20.6 1.0 2.0 2.5 7.5 12.5 25.0 area per 1 mL of blood (mm²/mL)

FIG. 16 shows a relationship between number of platelets and incubationtime. This reveals that the platelets are more significantly activatedwith the greater ratio of surface area of the glass processed body per 1ml of the blood to result in agglutination. Also, it was revealed that asample to which no glass processed body was added takes considerabletime, i.e., about 90 min, until blood agglutination was observed.Furthermore, provided that the glass processed body was not addedfollowed by leaving to stand for a long period of time to facilitaterelease of the growth factor from the platelets, the serum is oftencoagulated or a large amount of fibrin may be deposited in the followingprocess, due to insufficient activation of other coagulation factors.Thus, in the container of samples B to I according to ExperimentalExample, the platelets can be rapidly agglutinated because the glassprocessed body was stored, suggesting that growth factors derived fromthe platelets which will be required for preparation of the serum can bereleased at high efficiency.

Example 2 Determination of Growth Factor Releasing Effect

Study of effect on release of growth factor was conducted. Five samplesamong 9 samples used in Example 1 were selected, and fresh blood from 5subjects was added to each sample. Measurement of the growth factorafter 20 minutes elapsed is performed. Specifically, after incubationunder conditions shown in Table 2 below by the method that is similar toExample 1, amount of the growth factors (TGF-β1, PDGF-BB) was measuredby a commercially available test kit (manufactured by R&D SYSTEMS, Inc.)using a microplate reader (manufactured by Multiskan BICHROMATICLabsystem). The measurement was represented as a ratio to the amount inthe specimen having a contact area of 0 with the glass processed body.The results are illustrated in FIG. 17 and FIG. 18. In the Figures, theabscissa shows the surface area of the glass, and the ordinate shows theamount of each growth factor. As is clear from the FIG. 17, amount ofrelease of the growth factor was significantly increased when only aslight amount of the glass processed body, i.e., 0.6 mm² per 1 ml of theblood was added. However, it was demonstrated that the amount of TGF-β1achieves approximate equilibrium when the ratio of the surface area wasenormously increased. Furthermore, in FIG. 18, significant increase inthe growth factor was found by merely adding the glass processed body injust a slight amount. Moreover, it was found that the amount of PDGF-BBachieves approximately equilibrium when the ratio of the surface areawas enormously increased, similarly to FIG. 17.

TABLE 2 Sample A C E G I Diameter — 1 mm 4 mm Number 0 3 10 3 10 Ratioof glass surface area per 0 0.6 2.0 7.5 25.0 1 mL of blood (mm²/mL)

Example 3 Test of Hemolyzing Properties by Glass Processed Body

Examples 1 and 2 demonstrated that addition of the glass processed bodyis effective in activation of platelets and increase of growth factors.However, there was concern that addition of the glass processed body maycause hemolysis during preparation of the serum. Thus, the relationshipbetween the amount of addition of the glass processed body and hemolysiswas studied.

Prepared bovine blood was added to each of the samples A, F, H, and Iused in Example 1 and samples J to M prepared to have a wide range ofthe ratio of glass surface area per 1 ml of the blood, and incubatedwhile stirring. Sampling thereof was conducted in a time dependentmanner, and concentration of hemoglobin in the supernatant obtained bycentrifugal separation was measured (Hemoglobin B-test Wako,manufactured by Wako Pure Chemical Industries, Ltd.). Results followingthe incubation for 90 minutes are illustrated in FIG. 19. It wasdemonstrated that increase rate of hemoglobin in the supernatant iselevated as the surface area of the glass processed body stored insideof the blood reservoir is increased. This indicates that the glassprocessed body had an excessive contact area with the blood, andaccounted for disruption of erythrocytes (hemolysis) in the activationpromoting step or the centrifugal separation step very often. However,it was also demonstrated that the hemolyzing property that is equal tothe sample A without addition of the glass processed body was exhibitedup to 12.5 mm².

TABLE 3 Sample A F H I J K L M Number (4 mm diameter) 0 1 5 10 20 35 5075 Ratio of glass surface 0 2.5 12.5 25 50 87.5 125 187.5 area per 1 mLof blood (mm²/mL)

Example 4 Determination of Proliferation of Rat Stem Cell

Blood collected from a human was shaken for 20 minutes in a bloodreservoir in which any one of the aforementioned glass processed bodieswas stored, with the contact area of the glass processed body per 1 mlof the blood being adjusted to 0 mm² or 1.5 mm². The blood aftercompleting shaking was centrifuged (conditions of centrifugalseparation: 2250 g×10 min, 4° C.) to isolate the supernatant. Aftersubjecting the supernatant to a heat treatment at 56° C. for 30 minutes,the liquid was filtrated with a 0.22 μm filter, and preserved at −80° C.The supernatant was thawed upon cell culture, and added to a medium forcell culture. The cells subjected to this Example were cells derivedfrom bone marrow which were prepared by previously culturing cellsobtained from rat femur bone marrow to give adhesive cells. Tens ofthousands of cells per well were inoculated and cultured using a mediumto which the supernatant obtained by adding the glass processed, thesupernatant obtained without adding the glass processed body, or acommercially available fetal bovine serum for cell culture was added togive a concentration of 10%. On days 1, 3, and 7 after initiating theculture, the cell number was counted. Results obtained by determiningthe effects on cell proliferation are illustrated in FIG. 20.

As shown in FIG. 20, it is revealed that presence or absence of contactwith the glass processed body during preparation of the serum which isadded upon culture of the cells derived from rat bone marrow markedlyaffect the cell proliferation performance. Also, a more favorable resultwas obtained with the supernatant obtained by adding the glass processedbody, in comparison with the fetal bovine serum which had been generallyused hitherto.

Example 5 Recovery of Erythrocytes

Ascertainment of recovered erythrocytes was carried out. Contact area ofthe glass processed body was adjusted to be 12.5 mm² per 1 ml of theblood, and 20 ml of blood was collected, followed by shaking whilestirring with Multi Shaker MMS-300 (manufactured by Tokyo Rikakikai Co.,Ltd.) for 60 min. The blood collected in a test tube and left to standfor the same time period according to a conventional method ofpreparation of a serum was used as a control, and time dependentalteration of number of the erythrocytes was determined. The results areillustrated in FIG. 21. When number of the erythrocytes immediatelyafter collecting the blood was assumed to be 100%, about 80% of theerythrocytes remained after shaking with the glass processed body for 60min. On the other hand, when the serum was collected with a conventionalmethod, a large fraction of the blood in the container formed a clot,and thus only 10% of the erythrocytes were recovered in 60 minutes aftercollecting the blood.

Example 6 Recovery of Serum from Platelet-Rich Plasma (PRP)

Possibility of preparing a serum containing a large amount of growthfactors was verified also from the blood which had been collectedpreviously using an anticoagulant such as a CPD solution, or fromplatelet-rich plasma (PRP) prepared by apheresis. Fresh human blood towhich CPD was added to yield a final concentration of 12.2% wasprepared. This CPD-added blood was centrifuged under a condition of 760g, for 10 min (22° C.) to prepare platelet-rich plasma. Incubation of0.8 mL of thus resulting platelet-rich plasma in a vessal to whichcalcium chloride and glass processed body were previously added as shownin Table 4 was initiated at 37° C., and the container was shaken freely.The time period was measured which elapsed following the addition of theplatelet-rich plasma until the time point when fibrin was deposited fromthe platelet-rich plasma, resulting in lowering of fluidity inappearance. Each specimen with lowered fluidity was immediatelycentrifuged under a condition of 2,250 g, for 10 minutes (4° C.), andthe resulting supernatant was isolated. Thereafter, the amount ofPDGF-BB and the amount of TGF-β1 included in this supernatant weremeasured. Thus the measured amount of each growth factor is illustratedin FIG. 22 as a ratio (%) to the amount of each growth factor includedin the serum prepared from the identical blood.

TABLE 4 Specimen 1 2 3 Amount of added calcium chloride 0.01 mM Amountof added glass processed body 0 mm² 40 mm² 80 mm²

By adding the glass processed body in the amount of addition as shown inFIG. 22, or by increasing the area, coagulation time of theplatelet-rich plasma was shortened, and the amount of release of PDGF-BBand TGF-β1 was also increased.

Example 7 Determination of Effect of Addition of Air

Air or the glass processed body was added to the blood storage partunder the conditions shown in Table 5, respectively. To this bloodstorage part was charged 20 ml of fresh human blood, and incubated whilestirring with Multi Shaker MMS-300 (manufactured by Tokyo Rikakikai Co.,Ltd.). Number of platelets after 20 minutes elapsed was counted usingMultiparameter automated hematology analyzer K-4500 (manufactured bySYSMEX CORPORATION).

TABLE 5 Specimen 1 2 3 4 5 6 7 Total area of added glass 0 0 0 0 50 5050 processed body (mm²) Total quantity of added 0 2.5 5.0 20 2.5 5.0 20air (cc)

As shown in FIG. 23, in the specimen 1 prepared without adding eitherthe glass processed body or the air, approximately 90% of the plateletsremained even after a lapse of 20 minutes. When the air alone was added(specimens 2 to 4), residual ratio of the platelets decreased inproportion as the quantity of the added air. Additionally, when theglass processed body and the air were used in combination, decrease inresidual ratio of the platelets was remarkable, and proportionalcorrelation was found between the quantity of the added air and theresidual ratio of the platelets. Therefore, it was suggested that theair alone also exerted the effect of promoting the activation andagglutination of platelets although not comparative to the glassprocessed body, and that the air in combination with the glass processedbody achieves the effect to further promote the activation andcoagulation of the platelets.

The aforementioned Example 1 revealed that to store the glass processedbody as a blood coagulation accelerating substance in the bloodreservoir is effective for allowing the platelets to coagulate rapidly.More specifically, when the blood reservoir including the glassprocessed body stored therein is used, growth factors derived from theplatelets which will be necessary for preparing a serum from thecollected blood can be produced quickly with high efficiency. Therefore,when such a glass processed body is stored in the blood componentseparator, a large amount of serum can be quickly prepared (produced).

Moreover, Example 2 verified that growth factors that are useful in cellproliferation are sufficiently released during the preparation step intothe serum prepared from the blood collected in a container in which theglass processed body was stored therein, and consequently, addition ofthe serum to a medium contributes to proliferation of cells derived frombone marrow. Therefore, storage of the glass processed body in the bloodcomponent separator is advantageous in preparation of a serum that iseffective in promotion of cell proliferation.

Furthermore, Example 3 revealed that storage of excess glass processedbody in the blood reservoir is not desirable in light of amount ofhemoglobin released in the supernatant of the serum separated in thecentrifugal separation step, because such storage may lead to disruptionof erythrocytes (hemolysis) during shaking and centrifugal separation.

Additionally, Examples 4 to 7 showed that after preparing the serum thateffectively acts on cell cultures, a large fraction of erythrocytescould be recovered as individually isolated cells, not in the state of aclot. This suggests that autologous blood can be preserved as blood fortransfusion upon transplantation of stem cells to a patient with lowvolume of circulating blood or to a patient which receives transplantsurgery which may be accompanied by heavy bleeding. Hence, preparationof the serum under shaking using a container for collecting blood inwhich the glass processed body is stored also enables recovery ofcomponents other than serum.

As in the foregoing, in connection with the glass processed body in theblood reservoir taking into account Examples 1 to 7, it is proven thatthe surface area of the glass processed body to the volume of reservableblood in the container is desirably defined to have a relationship of0.1 mm²/ml or greater in light of promotion of the activation ofplatelets and coagulation factors and recovery of growth factors, and ispreferably defined to have a relationship of 25.0 mm²/ml or less also inlight of factors of occurrence of hemolysis.

INDUSTRIAL APPLICABILITY

As explained hereinabove, the blood component separator of the presentinvention can prepare a serum quickly and in a large amount in whichpropagation of microorganisms is suppressed. Thus, it is suited forpreparation of a large amount of serum which may be used for stem cellculture in regenerative medicine.

The invention claimed is:
 1. A blood component separation storageapparatus for separating a plurality of blood components of blood so asto be stored therein, the blood component separation storage apparatuscomprising: a blood reservoir for holding the blood; and a componentstorage part connected to the blood reservoir aseptically and in anair-tight manner, wherein: the blood reservoir contains an anticoagulantwhich suppresses coagulation of the blood; the component storage part isprovided with a plurality of bags connected together aseptically and inan air-tight manner; a part of the plurality of blood componentscontained in the blood in the blood reservoir is separated and stored ina first bag of the plurality of bags, the first bag containing a bloodcoagulation accelerating substance which is brought into contact withthe part of the blood components stored in the first bag and aneutralizing agent which neutralizes the anticoagulant; and the bloodcoagulation accelerating substance is insoluble in the blood and has aspherically shaped body made with a silicon dioxide compound, and has asurface area of 0.1 mm² to 25 mm² per 1 ml of blood.
 2. The bloodcomponent separation storage apparatus according to claim 1, whereinfractionation of a serum and other components is conducted in thecomponent storage part.
 3. The blood component separation storageapparatus according to claim 1, wherein the blood coagulationaccelerating substance is a granulated substance capable of activatingplatelets in the blood and of letting fiber in the blood adhere tosurfaces of the platelets.
 4. The blood component separation storageapparatus according to claim 1, wherein the blood coagulationaccelerating substance is a solid having a specific gravity greater thanthe blood, the blood coagulation accelerating substance settling outdownwardly so that a serum and coagulation factors are fractionated. 5.The blood component separation storage apparatus according to claim 1,wherein at least one kind selected from the group consisting of a CPDsolution and an ACD-A solution is contained as the anticoagulant.
 6. Theblood component separation storage apparatus according to claim 1,wherein the neutralizing agent contains at least calcium ions.